CN110075269B

CN110075269B - Application of Murabutide in preparation of medicine for preventing and treating bone marrow, small intestine and spleen injuries caused by ionizing radiation

Info

Publication number: CN110075269B
Application number: CN201910316873.6A
Authority: CN
Inventors: 刘蕾; 蔡建明; 高福; 杨彦勇; 曲红金; 崔建国; 廖泽彬; 曹堃; 陈媛媛; 刘哲
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2019-04-19
Filing date: 2019-04-19
Publication date: 2021-06-29
Anticipated expiration: 2039-04-19
Also published as: CN110075269A

Abstract

The invention relates to the field of new application of medicines, in particular to application of Murabutide in preparation of medicines for preventing and treating bone marrow, small intestine and spleen injuries caused by ionizing radiation. The Murabutide provided by the invention has the advantages of low toxic and side effects, obvious prevention and treatment effect on radiation-sensitive organ injury and the like when being used for preparing the medicine for preventing and treating the injury of bone marrow, small intestine and spleen caused by ionizing radiation. The method shows the unique advantages of Murabutide in preventing and treating ionizing radiation-induced multi-radiation-sensitive organ injury, and aims to explore a more efficient and low-toxic treatment and treatment method for preventing and treating ionizing radiation injury; at present, the effective prevention and treatment technical means for the multiple organ injury caused by ionizing radiation is still lacked at home and abroad, and the method is also one of the key problems which need to be researched and solved urgently in the fields of radiology and clinical radiotherapy. Therefore, Murabutide has wide development and application prospects in the medical field of China as a medicine for preventing and treating the damage of multiple organs of the whole body caused by ionizing radiation.

Description

Application of Murabutide in preparation of medicine for preventing and treating bone marrow, small intestine and spleen injuries caused by ionizing radiation

Technical Field

The invention relates to the field of new application of medicines, in particular to application of Murabutide in preparing medicines for preventing and treating bone marrow, small intestine and spleen injuries caused by ionizing radiation.

Background

With the rapid development of science and technology, nuclear energy has increasingly become an important component of scientific research and military strength of various countries, and although nuclear energy management is improved day by day, the possibility of serious accidents and the leakage of a large amount of radioactive substances in a nuclear power plant still exists. According to incomplete statistics, the number of radioactive accidents in China from 1954 to 2015 2007 is more than 20 per year on average, and radiotherapy, as an important auxiliary means for clinical malignant tumor treatment, inevitably damages normal tissues around tumors while killing malignant tumor tissues, and causes sequelae of hematopoietic systems, digestive systems, immune systems and the like. The main reason why ionizing radiation causes body damage is that ionizing radiation causes DNA fragmentation by direct and indirect action, and then hydroxyl radicals generated by water molecules decomposing under the action of radiation cause secondary damage to each organ, while unrepaired DNA further aggravates the damage. The acute injury of the organism caused by ionizing radiation is mainly reflected in bone marrow, intestinal tracts, spleen and the like and is represented as follows: after radiation, the hematopoietic function of bone marrow is sharply reduced, the functions of hematopoietic stem cells and hematopoietic progenitor cells are damaged, and the whole blood cells are reduced; after the small intestine is irradiated, intestinal villi are broken and fractured, the number of intestinal crypts is also obviously reduced, and inflammatory cells are increased and infiltrated; the boundary between the white marrow and the red marrow in the spleen becomes fuzzy, the area of the white marrow is reduced, the small spleen body is atrophied, the structure and the function of each organ are damaged, and simultaneously, the disturbance of immune balance is also accompanied, so the damage of ionizing radiation to the organism is further aggravated. Therefore, the urgency of the study is self evident in acute radiation diseases caused by nuclear leakage accidents or by side effects of clinical radiotherapy. At present, the international prevention and treatment of acute radiation diseases are mainly limited to hematopoietic stem cell transplantation, drug therapy and symptomatic support therapy, but the effect is poor, so that the prevention and treatment measures for the acute radiation diseases need further research and exploration. Researches show that Toll-like receptors (TLRs) have a certain protective effect in acute radiation protection, but the TLRs have the defects of limited distribution range and large toxicity when being used excessively, so that the practical application of the TLRs in clinic is limited, and further research on other efficient and low-toxicity radiation protection measures is urgently needed.

Pattern Recognition Receptors (PRRs) have been of widespread interest as important means of reducing radiotoxicity, and include mainly Toll-like receptors (TLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), and the like. TLRs have been studied to activate TLRs to exert radioprotective effects by recognizing microbial ligands on the host cell surface or endosomes, unlike TLRs, NLRs recognize bacterial products located in the host cytoplasm and activate defense. NLRs comprise NOD1 and NOD2, after NOD2 is activated, nuclear factor NF-kB signal path and MAPK signal path are induced to be activated, the two signal paths are proved to be used as radiation protection paths for regulating cell proliferation and apoptosis, and in addition, the activation of NOD2 has the functions of stimulating hematopoietic function recovery and enhancing the immunity of the organism. Whereas NOD2 and TLRs have been shown to interact in host defense and microbial recognition, activation of NOD2 is likely to be an effective radioprotection strategy.

Muramyl Dipeptide (MDP) is a well-known NOD2 agonist, however, due to the high pyrogenicity and arthritic formation of this molecule, a safe MDP derivative Murabutide (abbreviated as MBD) was developed in the later stages. The research shows that MBD as a clinically approved immunomodulator can improve the biological activity of cytokines and chemokines in immunotherapy research of HIV patients, and when the MBD is formulated with other adjuvants, the MBD is also a promising immunoadjuvant for resisting tuberculosis and anthrax infection, and the safety of the MBD is clinically verified. However, the protective effect of MBD on ionizing radiation has not been explored. At present, the research on ionizing radiation prevention and treatment is mostly in a basic stage, and the period from research and development to formal clinical use is long and the cost is high, so that the clinical transformation period can be effectively shortened by re-research and development and application of the existing clinical drugs, and the method is also one of effective means for developing novel radioprotectors in the medical field.

MBD is used as an agonist of NOD2, has obvious effect of preventing and treating bone marrow, small intestine and spleen injuries caused by acute systemic radiation, and has the definite advantage of low toxicity. No report on the prevention and treatment effects of an agonist MBD of NOD2 on the acute systemic radiation-induced bone marrow, small intestine and spleen injuries is found in the prior art.

Disclosure of Invention

The invention aims to provide a new application of NOD2 agonist Murabutide (MBD for short), namely the application in preparing medicines for preventing and treating acute bone marrow, small intestine and spleen injuries caused by ionizing radiation.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, the application of Murabutide in preparing a medicine for preventing and treating bone marrow, small intestine and spleen injuries caused by ionizing radiation is provided. The structural formula of Murabutide is shown as the following formula I:

furthermore, the Murabutide can prevent and treat the injury of bone marrow, small intestine and spleen caused by ionizing radiation by activating NOD 2.

Furthermore, the medicine is used for relieving hematopoiesis inhibition after ionizing radiation and relieving the process of hematopoiesis cytopenia caused by ionizing radiation.

Furthermore, the medicine is a medicine for inhibiting acute ionizing radiation damage of small intestine villi and small intestine crypts.

Furthermore, the medicine is used for relieving spleen white marrow damage caused by ionizing radiation.

Furthermore, the medicine is a medicine for reducing the apoptosis of small intestine and spleen cells caused by ionizing radiation.

Further, the medicament is a medicament for enhancing NOD2 rise caused by ionizing radiation.

Further, the medicament is a medicament for enhancing cell proliferation and independent survival capability after ionizing radiation.

Furthermore, the medicine is used for reducing double-stranded DNA damage caused by ionizing radiation.

Further, the ionizing radiation is⁶⁰And (4) irradiating Co gamma rays.

Further, the dose of Murabutide administered in the medicament is 15mg/kg, and the Murabutide is administered 2 hours before ionizing radiation.

In a second aspect of the present invention, there is provided an ionizing radiation protective drug, wherein the active ingredient of the ionizing radiation protective drug is Murabutide.

Further, the ionizing radiation protective medicine is an injection.

Furthermore, the ionizing radiation protection medicament also comprises pharmaceutically acceptable auxiliary materials.

The present invention used C57BL/6 male 8 week old mice, ordered from the laboratory animals center at the second university of military medicine, for animal experiments. The mice are raised in an environment with 12-hour light and shade cycle, 20-25 ℃ and free eating of sterilized water and grain. The mice were then divided into 3 groups: group 1, non-irradiated + PBS control group, second group, irradiated + PBS group, 3 group, irradiated + MBD group. Mice receiving 7Gy ⁶⁰2 hours before the Co gamma-ray total body irradiation, the mice were administered with intraperitoneal injection of PBS or MBD, and the survival of the mice was recorded every day within 30 days after the irradiation. The results found that MBD pretreatment significantly improved survival of mice after irradiation. Meanwhile, bone marrow, small intestine and spleen of 3 groups of mice were taken 0, 1 and 3 days after irradiation, respectively, fixed, embedded in wax block, sectioned and HE-stained. As a result, it was found that the number of nucleated cells in the bone marrow was normal in the non-irradiated group and that there was no abnormality in hematopoietic function. After 1 day, the number of nucleated cells in the bone marrow of the irradiated group is reduced, and after 3 days, the number of the nucleated cells in the bone marrow is continuously reduced, so that the marrow is vacuumed, and the hematopoietic function of the bone marrow is obviously inhibited. By comparing the irradiated group with the MBD group administered before irradiation, it was found that the MBD group had early hematopoietic suppression after irradiationObviously relieves the hematopoietic cell reduction degree. Meanwhile, the HE slice of the small intestine shows that the villi of the small intestine of the non-irradiated group have complete structure, close interval and normal villi length, the villi are loosened and disordered and even broken after irradiation, the number of crypts of the small intestine is reduced, but the MBD pretreatment can obviously inhibit the acute radiation damage of the villi and the crypts of the small intestine after irradiation. In the HE section of the spleen tissue, the boundary between the white marrow and the red marrow becomes fuzzy on the 1 st day after irradiation, the white marrow area is reduced and is increased along with the time development, while the red and white marrow boundary of the MBD pretreatment group is relatively clear and the reduction of the white marrow area is not obvious, so that the MBD can reduce the damage of the radiation to the spleen to a certain extent. Meanwhile, the invention also detects the apoptosis condition in small intestine and spleen tissues after the 1 st day of radiation, and finds that the MBD pretreatment can obviously reduce the apoptosis of the small intestine and spleen tissues caused by radiation.

In addition, the invention uses Western blot to detect NOD2 protein of human normal small intestine epithelial cell HIEC. It was found that MBD was able to significantly activate the expression of NOD2,⁶⁰co gamma irradiation can also activate the expression of NOD2, and the addition of MBD before irradiation can significantly enhance the irradiation-induced increase of NOD 2. In addition, the invention also provides different concentrations (all of which are)<5 mug/mL) of MBD in PBS, adding the dissolved MBD into HIEC, and determining the toxic concentration of MBD by detecting the growth of cells after 24 hours of culture by CCK-8 method<No significant cytotoxicity exists in the concentration range of 5 mu g/mL. And, after MBD of various concentrations was added to HIEC cells 2 hours before irradiation, 8Gy was administered to the cells⁶⁰Co gamma ray irradiation, and CCK-8 method detection after 24 hours finds that MBD pretreatment can obviously enhance the proliferation capacity of the irradiated cells compared with the irradiated group, and particularly the MBD pretreatment group with 0.5 mu g/mL and 1 mu g/mL is obvious. Subsequently, the HIEC cells were treated with MBD at 1. mu.g/mL in the present invention, and then the cells were administered with different doses (0, 2, 4, 6, 8Gy)⁶⁰Co gamma ray irradiation and cell growth and proliferation detection by a clone formation rate method show that after MBD pretreatment, the proliferation and independent survival capability of HIEC cells after irradiation can be obviously enhanced along with the increase of irradiation dose. Meanwhile, the invention adopts the comet electrophoresis method to detectOf 8Gy⁶⁰The degree of damage to double-stranded DNA of HIEC cells after Co gamma ray irradiation, and the result shows that compared with the irradiation group, the MBD pretreatment group can obviously reduce the damage to double-stranded DNA caused by ionizing radiation.

Therefore, the invention claims the application of MBD in preparing medicines for preventing and treating bone marrow, small intestine and spleen injuries caused by ionizing radiation. Under the concentration of the medicine without any obvious effect on animals and cells, normal cells of animals and human are pretreated by MBD before receiving ionizing radiation, so that the damage of the ionizing radiation can be reduced, and the protective effect of the ionizing radiation is exerted.

Compared with the prior art, the invention has the following advantages:

the Murabutide provided by the invention has the advantages of low toxic and side effects, obvious prevention and treatment effects on the damage of radiation-sensitive organs (bone marrow, small intestine and spleen) and the like when being used for preparing the medicine for preventing and treating the damage of bone marrow, small intestine and spleen caused by ionizing radiation. The performance shows that Murabutide has very wide research prospect in the fields of biological medicine and clinical treatment as a medicine for preventing and treating the injury of bone marrow, small intestine and spleen caused by ionizing radiation, has unique advantages in preventing and treating the injury of multiple radiation sensitive organs caused by ionizing radiation and aims to explore a more efficient and low-toxic method for treating and treating the injury of ionizing radiation; at present, the effective prevention and treatment technical means for the multiple organ injury caused by ionizing radiation is still lacked at home and abroad, and the method is also one of the key problems which need to be researched and solved urgently in the fields of radiology and clinical radiotherapy. Therefore, Murabutide has wide development and application prospects in the medical field of China as a medicine for preventing and treating the damage of multiple organs of the whole body caused by ionizing radiation.

Drawings

FIG. 1 shows 7Gy of the present invention⁶⁰A contrast graph of survival curves of mice in a control group, an irradiation group and an MBD administration group before irradiation after the total body irradiation of the Co gamma rays;

FIG. 2 shows 7Gy of the present invention⁶⁰Comparing the HE slices of the mouse bone marrow of the control group, the irradiation group and the MBD administration group before irradiation after the total body irradiation of the Co gamma rays;

FIG. 3 shows 7Gy of the present invention⁶⁰Co gamma-ray irradiation group after whole body irradiation control group, irradiation group and MBD administration group before irradiation mouse small intestineHE slice contrast map;

FIG. 4 shows 7Gy of the present invention⁶⁰Comparing HE slices of spleen of mice in a control group, an irradiation group and an MBD administration group after total body irradiation of Co gamma rays;

FIG. 5 shows 7Gy of the present invention⁶⁰Apoptosis changes in small intestine and spleen in the control group irradiated with Co gamma rays after total body irradiation, the irradiated group and the MBD-administered group before irradiation;

FIG. 6 is a graph showing the toxicity studies of MBD at various concentrations of the present invention and the detection of activated protein expression of NOD2 after the addition of MBD;

FIG. 7 shows the present invention⁶⁰Modification of growth and proliferation of HIEC cells by MBD pretreatment after Co gamma ray irradiation;

FIG. 8 is a diagram of 8Gy of the present invention⁶⁰MBD pretreatment after Co gamma irradiation changes the extent of DNA damage after HIEC cell irradiation.

Detailed Description

The invention is further described below with reference to the following figures and examples:

materials: cell lines and cell cultures: human normal intestinal epithelial cells HIEC (purchased at fourth university of military medicine) were cultured in RMPI 1640 containing 10% fetal bovine serum at 37 ℃ under 5% CO₂Culturing in an incubator.

Drugs and primary agents: the drug Murabutide (abbreviated as MBD) purchased from INVIVOGEN, USA; RMPI 1640 medium, fetal bovine serum and pancreatin were purchased from Gibco; the CCK-8 kit is purchased from the institute of Homon chemistry, Japan; the crystal violet stain, the molecular weight of standard protein, SDS-PAGE sample buffer, RIPA protein lysate, 30% Acry-Bis, Tris-HCl, Ammonium Persulfate (AP), SDS, Tetramethylethylenediamine (TEMED) and Propidium Iodide (PI) stain were purchased from Jiangsu Bitian institute of biotechnology; the tissue fixing solution, the TE electrophoresis buffer solution, the membrane transferring solution and the TBST washing solution are purchased from Wuhan Google Biotech limited company; TUNEL kit purchased from roche diagnostics products ltd; NOD2(CARD15) antibody, GAPDH antibody were purchased from Abcam.

Mice: c57BL/6 male 8 week old mice were ordered by the laboratory animal center at the second university of military medicine.

Wherein, the irradiation conditions are as follows: radiation center (second)Navy medical college of military medical university, Shanghai, China) of⁶⁰C gamma ray irradiation. All the irradiated animals receive single dose of 7Gy, the dose rate is 1Gy/min, and the irradiation is whole body irradiation; the irradiated cells received a single dose of 8Gy at a dose rate of 1 Gy/min.

Statistical treatment: all experiments in the following examples were repeated 3 times or more, and the results were expressed as Mean ± SD. Relevant data were subjected to t-test using GraphPad Prism6 statistical software with significant differences of P < 0.05.

Example 1:

firstly, a mouse model of ionizing radiation induced systemic injury is established, and 30 male C57BL/6 mice with the age of 8 weeks are randomly divided into three groups: 10 control groups, 10 irradiation groups and 10 pre-irradiation MBD administration groups (7Gy + MBD) were used⁶⁰The mice were irradiated systemically with a single dose of 7Gy of Co gamma radiation. MBD (15mg/kg MBD +0.2mL PBS/mouse) or PBS (0.2 mL/mouse) was administered by intraperitoneal injection 2 hours before irradiation. Mice (control group, irradiated group and pre-irradiated MBD-administered group) were observed and recorded regularly daily for 30 days after irradiation. As shown in fig. 1, the mice in the irradiated group began to die and showed signs of body slimming and decreased appetite starting from 7 days after irradiation, while the death time in the MBD-treated group before irradiation was significantly delayed. In combination, MBD pretreatment can significantly prolong the survival time of mice after ionizing radiation.

Example 2:

(1) establishing a whole body injury mouse model caused by ionizing radiation as in example 1;

(2) the mice were divided into groups, administered and irradiated in the same manner as in example 1, and the mice were sacrificed at different time points (0 day, 1 day, 3 days) after irradiation, femoral bone tissue, small intestine tissue and spleen tissue were taken, fixed, embedded in wax block, and sectioned for HE staining. The HE results of the femoral tissues are shown in fig. 2, and compared with the irradiation control group, the irradiation group has obviously empty bone marrow cavities and reduced nucleated cell number, which indicates that hematopoietic cells are reduced; while the bone marrow cavity of mice treated with MBD before irradiation (irradiation + MBD group) was relatively full, so that the decrease of hematopoietic cells and the suppression of hematopoietic function caused by ionizing radiation were relieved to a certain extent. The HE results of the small intestine are shown in fig. 3, the intestinal villus of the irradiated mice is disordered, even broken and fallen off, and the number of intestinal crypts is reduced compared with the irradiated control group, and statistics show that the intestinal villus length and the number of crypts of the irradiated group intervened by MBD before irradiation are higher than those of the single irradiated group. Meanwhile, HE staining of the spleen was also detected in the present invention, as shown in fig. 4, as the time after irradiation was prolonged, the junction between white marrow and red marrow in the irradiated group was blurred and gradually increased, and the area of white marrow was also significantly reduced, suggesting impaired immune function. As can be seen from the statistical chart, treatment with MBD before irradiation can reduce the white marrow damage caused by irradiation, which indicates that the spleen has radiation protection effect.

Example 3:

(2) the small intestine and spleen wax masses 1 day after irradiation were collected from each group of mice in example 2, sectioned and subjected to TUNEL staining, and the number of cells positive to TUNEL was used to indicate apoptosis after irradiation. As shown in FIG. 5, the number of apoptotic cells in the non-irradiated group of small intestine and spleen was very small, and the apoptotic expression was normal cell metabolism. The number of TUNEL positive cells in the irradiated group was significantly increased after irradiation, while MBD pretreatment before irradiation significantly reduced irradiation induced apoptosis.

Example 4:

(1) cell culture: 10% fetal bovine serum was previously added to the RMPI 1640 medium, and HIEC cells were cultured in the medium. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator, carrying out passage once when the cells grow to 80% -90%, and taking the cells in the logarithmic growth phase for subsequent experiments.

(2) The CCK-8 method is used for detecting MBD toxicity: the HIEC cells in good condition were seeded in a 96-well plate (3X 10)⁵One/well), 8 parallel control wells were set for each concentration. After the cells are attached to the wall completely, MBD with different concentrations is added to make the final concentrations respectively 0, 0.025, 0.25, 2.5 and 5 mug/mL, and each well of the culture medium is 100 mug L. After 24 hours of incubation, 10. mu.L of CCK-8 solution was added to each well, and incubation was continued for 1 to 3 hours in an incubator, and absorbance (OD value) at 450nm was measured using a microplate reader. Finally, the survival rate of the cells is multiplied by 100 percent compared with the OD value of the drug adding group/the OD value of the control groupThe survival rate of the MBD group cells with different concentrations is calculated by the formula (2). As shown in FIG. 6, no inhibition of cell growth and proliferation was observed up to a maximum concentration of 5. mu.g/mL of MBD.

(3) Detecting NOD2 protein expression by Western blot electrophoresis: the HIEC cells with good growth state are plated to ensure that the cells are completely attached to the wall in 8Gy⁶⁰MBD with final concentration of 1 mug/mL and 5 mug/mL is added 2 hours before Co gamma ray irradiation, protein is extracted 0.5 hour after irradiation, and Western blot electrophoresis is carried out to detect the expression condition of NOD2 protein. As shown in FIG. 6, while MBD at a concentration of 1. mu.g/mL significantly activated the expression of NOD2 in HIEC cells, and irradiation also activated NOD2, resulting in an increase in expression, the addition of MBD before irradiation significantly enhanced the increase in NOD2 due to irradiation, as compared with the case of the single irradiation group.

Example 5:

(1) the cells were cultured as in example 4;

(2) the CCK-8 method is used for detecting the cell proliferation capacity: the HIEC cells are inoculated to the adherent wall by using the method and are given 8Gy⁶⁰MBD with different concentrations is added into each parallel group 2 hours before Co gamma ray irradiation, and after 24 hours of culture, the proliferation condition of cells in different concentration groups is detected by a CCK-8 method. As can be seen in FIG. 7, compared with the irradiated group, the cell proliferation capacity of the MBD added before irradiation is stronger, and basically enhanced along with the increase of the administration concentration of the MBD in the concentration range of 1 mug/mL, which indicates that the MBD can relieve the inhibition of ionizing radiation on the proliferation capacity of the cells and promote the cell proliferation in the safe and nontoxic concentration range.

(3) Cell clone formation assay for cell viability: the HIEC cells in good growth state are inoculated into a 6-well plate, and different cell numbers are inoculated according to the subsequent irradiation dose to be received: 0. the inoculum sizes of 2, 4, 6 and 8Gy irradiation doses are 250, 500, 1000, 2000 and 4000 respectively. After the cells are completely attached to the wall, the irradiation group and the MBD group are added with 1 mu g/mL of MBD for treatment for 2 hours, and the irradiation group is added with PBS with the same amount as the negative control. After the cells were irradiated, the cells were cultured for about 10 days until cell colony formation (cell number > 50) was achieved, the medium was discarded, washed once with PBS, fixed with tissue fixative for 30 minutes, stained with crystal violet stain for 30 minutes, washed and naturally air-dried. Counting the clone colonies under a microscope, setting 3 multiple wells for each group to obtain an average value, and calculating the clone formation rate of the cells with different irradiation doses by adopting the clone formation rate (the number of the cell colonies formed by the irradiation dosing group/the number of the plated cells of the irradiation dosing group)/(the number of the cell colonies formed by the irradiation group/the number of the plated cells of the irradiation group) for each irradiation dose. As shown in fig. 7, MBD can significantly reduce the cell death rate of HIEC cells caused by ionizing radiation as the irradiation dose is increased.

Example 6:

(1) the cells were cultured as in example 4;

(2) detection of DNA damage by comet electrophoresis: before irradiation, the clean glass slide is immersed into the molten 1% high-melting-point adhesive and the back of the glass slide is wiped dry, so that the comet-star electrophoresis negative film is prepared. HIEC cells in good growth state were seeded in 6-well plates to a cell density of 3X 10⁵Per well. After the cells were completely adherent, MBD was added to the irradiation + MBD group 2 hours before irradiation to a final concentration of 1. mu.g/mL, and PBS was added to the irradiation group in equal amounts. Administration of 8Gy to cells ⁶⁰4 hours after Co gamma irradiation, each group of cells was collected and treated with Ca-free medium²⁺、Mg²⁺Washed once with PBS and washed with Ca-free buffer²⁺、Mg²⁺The PBS was blown into a single cell suspension to a cell density of 2X 10⁴and/mL, soaking 0.4mL of the single cell suspension into 1.2mL of 0.65% low-melting-point glue, uniformly mixing, and quickly spreading 0.5mL of the mixed solution on the surface of the bottom sheet of the glass slide. After air drying, the slide is immersed in a neutral cell lysate precooled at 4 ℃, is subjected to dark lysis overnight, is taken out and is placed in a horizontal electrophoresis tank, and is subjected to 25V electrophoresis for 25 minutes. The cells were washed with double distilled water, stained with 10. mu.g/mL PI for 20 minutes, washed with double distilled water, observed with a fluorescence microscope, and analyzed using CASP 1.2.3b2 software. As shown in FIG. 8, the ratio of the tail-to-total comet fluorescence and tail moment of the irradiated group were significantly increased, suggesting that the irradiation induced DNA damage, while the pretreatment with MBD before irradiation significantly reduced the irradiation induced DNA damage with statistical differences.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Claims

The Murabutide is applied to the preparation of medicines for preventing and treating injuries of bone marrow, small intestine and spleen caused by ionizing radiation; said ionizing radiation is⁶⁰Irradiating with Co gamma rays; the administration dose of Murabutide in the medicament is 15mg/kg, and the Murabutide is administered 2 hours before ionizing radiation.
2. The use of Murabutide according to claim 1 in the preparation of a medicament for the prevention or treatment of bone marrow, small intestine and spleen damage caused by ionizing radiation, wherein said medicament is a medicament for alleviating the hematopoietic inhibitory effect following ionizing radiation and alleviating the progression of hematopoietic cytopenia caused by ionizing radiation.
3. The use of Murabutide according to claim 1 in the preparation of a medicament for preventing and treating ionizing radiation-induced damage to bone marrow, small intestine and spleen, wherein the medicament is a medicament for inhibiting acute ionizing radiation damage to villi and crypts of small intestine.
4. The use of Murabutide in the preparation of a medicament for preventing and treating bone marrow, small intestine and spleen injury caused by ionizing radiation according to claim 1, wherein the medicament is a medicament for reducing spleen white marrow injury caused by ionizing radiation.
5. The use of Murabutide according to claim 1 in the preparation of a medicament for preventing and treating bone marrow, small intestine and spleen damage caused by ionizing radiation, wherein the medicament is a medicament for reducing apoptosis of small intestine and spleen cells caused by ionizing radiation.
6. The use of Murabutide according to claim 1 in the preparation of a medicament for preventing and treating bone marrow, small intestine and spleen injuries caused by ionizing radiation, wherein the medicament is a medicament for enhancing the increase of NOD2 caused by ionizing radiation.
7. The use of Murabutide according to claim 1 in the preparation of a medicament for the prevention and treatment of bone marrow, small intestine and spleen damage caused by ionizing radiation, wherein said medicament is a medicament for enhancing cell proliferation and independent survival after ionizing radiation.
8. The use of Murabutide according to claim 1 in the preparation of a medicament for preventing and treating bone marrow, small intestine and spleen damage caused by ionizing radiation, wherein the medicament is a medicament for reducing double-stranded DNA damage caused by ionizing radiation.